Construction and immunoscreening of cDNA library of Echinococcus granulosus

Abstract

Objective To construct the λ ZAP Ⅱ cDNA library of Echinococcus granulosus for screening of immunodiagnostic candidate molecules.Methods The protoscoleces of Echinococcus granulosus (E.granulosus) were isolated from the fertile cysts of E.granulosus infected sheep,and total RNA of the protoscoleces was extracted using Trizol reagent,then mRNA was purified from the total RNA using mRNA purification kit.cDNA was synthesized using cDNA synthesis kit with the purified mRNA as template,then blunt-ended,linked with EcoR Ⅰ adapter,phosphorylated,and cut by Xho Ⅰ enzyme using the λ ZAP Ⅱ EcoR I/CIAP-kit.The cDNA obtained was then separated by Sepharose CL-2B colum,and cDNA longer than 500 bp was collected and cloned into λ ZAP Ⅱ vector.The recombinant vectors were packaged into λ ZAP Ⅱ phage thus to constitute cDNA library.The cDNA library was immunoscreened by mixed patient sera of cystic echinococcosis and the positive clones obtained were sequenced and analysed by Blast module in NCBI and other informatics sofiwares.Results The λ ZAP Ⅱ cDNA library was constructed with the titer of 18 × 105plaque forming unit(pfu) /ml,the length range of the inserts 1.0 ～4.0 kb,the average length of 2.6 kb,and the insert efficiency of 93.8％. Five positive clones were obtained through immunoscreening of the cDNA library,and 2 of them are new genes of E.granulosus with multiple B cell epitopes.Conclusion The λ ZAP Ⅱ cDNA library of E. granulosus was successfully constructed and positive clones were obtained through immunoscreening,among them 2 clones are new genes which could become candidate molecules for diagnosing cystic echinococcosis. Key words: Echinococcus granulosus; Protoscolex; cDNA library; Immunoscreening